Introduction

Lung cancer is the primary cause of cancer-related death in both men and women with an overall 5-year survival rate of 15% (1, 2). Current first-line therapies often involve chemotherapeutic combinations that target the heterogeneity of signaling pathways active among malignant cell populations. Nevertheless, chemoresistance of tumor cells continues to pose a significant challenge for these strategies to be efficacious in prolonging patient survival. Hence, multimodal therapies that also stimulate antitumor immune responses are essential for long-term management of this disease (3).

Oxidative stress, resulting from elevated reactive oxygen species (ROS), is implicated in the initiation and progression of lung cancer (4). The host protective antitumor immunity is also often suppressed by several nonmalignant leukocyte lineages in the tumor microenvironment (TME), which significantly dampen the long-term efficacy of existing combination therapies for lung cancer (5, 6). Myeloid-derived suppressor cells (MDSC) generate ROS, associated free radicals, and immunoregulatory cytokines to suppress host CD4+ and CD8+ T-cell responses, thereby promoting tumor progression and metastasis (7–9). In patients with advanced stages of lung cancer, increased numbers of circulating MDSC correlate with immunosuppression and enhanced tumor burden (10). Despite the documented roles of these immunosuppressive cells in lung cancer, few immunomodulatory therapies have been successful at targeting MDSC to enhance long-term immunity. Development of treatment resistance following gemcitabine (Gem) therapy and elevation of ROS in the TME may impact infiltrating immune effector function, induce MDSC proliferation, and impair memory response, which is a critical component of protective immunity. We hypothesized, therefore, that a timely uncoupling of the MDSC-induced immunosuppression in the TME and ROS would not only enhance effects of gemcitabine, but also reactivate host antitumor immunity including both effector and memory functions and significantly enhance survival outcomes.

Herein, we report an innovative therapeutic strategy that both impairs MDSC function and induces effective memory T-cell responses against lung tumors. Using a syngeneic lung cancer mouse model, we demonstrate that gemcitabine, a well-established chemotherapy agent for lung cancer and a known inhibitor of MDSC expansion, along with superoxide dismutase mimetic (SOD mim), reduced tumor growth and resulted in long-term immunity and survival. This combination therapy enhanced the quantity and quality of the effector and memory CD8+ T-cell responses with an enrichment of self-renewing memory cells. Cellular redox–mediated regulation of STAT-3 activation and metabolism of the central memory and effector memory CD8+ T cells contributed to the long-term immunity. Furthermore, adoptive transfer experiments demonstrated that inhibition of MDSC and MDSC-generated ROS enhanced the persistence of memory CD8+ T cells, as well as their vigorous activity in response to reencountering tumor antigens as in stages of relapse. This study clearly delineates the role of MDSC in lung cancer progression and demonstrates utility of this combination therapy for inhibiting tumor and MDSC expansion, abrogating their key molecular functions/pathways and offers a new strategy for treatment of early lung cancer and prevention of relapse.

In vivo treatment regimen

LLC-challenged mice were treated with gemcitabine and a SOD mim either individually or in combination (see the treatment model in Fig. 2). Five days after tumor challenge via an intravenous route and 3 days after tumor challenge via an intracardiac route, mice were injected intraperitoneally with either PBS or 60 mg/kg gemcitabine (Sigma-Aldrich) in 50 μL/mouse and 10 mg/kg SOD mim [MnTE-2-PyP5+ (manganese (III) mesotetrakis (di-N-diethylimidazole) porphyrin], generously provided by Dr. James Crapo (National Jewish Hospital, Denver, CO), in 100 μL/mouse, twice in the first week and once in the following week. Alternatively, LLC-challenged mice received anti-Gr1 antibody (250 μg/100 μL; ref. 11) or immunoglobulin G (IgG) control (BioXCell) intraperitoneally at day 5 either individually or in combination with SOD mim. Catalase-deficient mice (a kind gift from Dr. Jaroslaw Zmijewski, The University of Alabama at Birmingham, Birmingham, AL) were also challenged with tumor and treated as described above.

Isolation of immune cells and FACS analysis

Infiltrating leukocytes were isolated from minced tumor and lung tissues from LLC-challenged mice by incubation in DMEM containing collagenase-B (2 mg/mL; Roche) and DNase I (0.02 mg/mL; Sigma Chemical) at 37°C for 30 minutes. This was followed by the addition of an equal volume of complete DMEM containing 10% FBS (see Supplementary Methods for additional details). ROS+ cells in tumor tissues were detected by flow cytometry as described before (12). To quantitate the percentage of apoptotic cells, CD8+ T cells from the tumor tissue were stained by a FITC–Annexin V Apoptosis Detection Kit (BD Biosciences). A total of 50 × 103 events were collected for all analyses using the BD LSRII cytometer (BD Biosciences) and the data were analyzed using FlowJo software.

Immunofluorescence microscopy

Detailed procedures for detection of MDSC and CD8+ T cells in snap-frozen tumor tissues are provided in the Supplementary Methods.

Cytotoxicity assay

CD8+ T-cell memory subsets and effector cells were isolated from LLC-challenged mice with gemcitabine and SOD mim treatment as reviewed previously and then used as the effector cells (E). LLC cells were used as the target population (T). The assay was set up with E:T ratios of 5:1, 10:1, 20:1, and 40:1. The cytotoxicity assay was performed using the LIVE/DEAD Cell-Mediated Cytotoxicity Kit (Molecular Probes). DiOC18+PI+ cells were identified as dead target cells and DiOC18+PI− cells were identified as live target cells by flow cytometry analysis. The percentage of cytotoxicity was calculated following the manufacturer's guidelines as [(Dead Cells/Live Cells)+effectors − (Dead Cells/Live Cells)−effectors] × 100.

Detection of thiols

Cell surface thiols on CD8+ T cells from tumor tissues of LLC-challenged mice treated with gemcitabine and SOD mim were detected by staining with 5 μmol/L Alexa Fluor 633–coupled maleimeide (ALM-633; Life Technologies) for 15 minutes on ice, washed with PBS, and analyzed by flow cytometry.

Metabolomic analysis

CD8+ T-cell memory cell subsets were immunopurified from tumor tissues of LLC-challenged mice following treatment as described above. Cells (100 mg wet weight) were rinsed with ice-cold, isotonic PBS twice and the cell pellet was resuspended in cold methanol (−45°C in dry ice). The methanol slurry was then transferred to a cooled centrifuged tube and centrifuged at 14,000 rpm at −20°C for 10 minutes. The methanol extracts were evaporated to dryness, derivatized, and reconstituted in 100 μL of water before analysis. Metabolites that contain an aldehyde or a ketone in their structure were derivatized using Amplifex (Applied Biosystems). Liquid chromatography/mass spectrometry-multiple reaction monitoring (LC/MS-MRM) analysis was then carried out with the mass spectrometer operating in both the positive and negative modes. Addition of Amplifex adds an amine group to the carbonyl of the ketone/aldehyde, which increases the mass of the compound by 115 amu. The LC system was reverse-phase with the mobile phase A of 0.1% HCOOH in water and mobile phase B of methanol + 0.1% HCOOH. A Synergi Hydro-RP C18 column, 2 mm × 250 mm was used for analysis.

Phospho-STAT3 flow cytometry and ELISA

Detailed procedures for detection and quantitation of p-STAT-3 activation in CD8+ T-cell memory cell subsets are provided in the Supplementary Methods.

Isolation of CD8+ T-cell memory populations and adoptive transfer

Detailed procedures for characterization, purification, and in vitro expansion of CD8+ T-cell memory populations are provided in the Supplementary Methods.

Statistical analysis

Data are represented as mean ± SD. One-way ANOVA with Tukey multiple comparison posttest and the Student t test was used for statistical comparisons using GraphPad Prism 5. Statistical significance was determined at the <0.05 level (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). Kaplan–Meier method was used for evaluation of survival patterns in tumor-bearing mice. Results were ranked according to the Mantel–Cox log-rank test and P < 0.05 was considered statistically significant. Survival was defined as time until death of mice due to excess tumor burden.

Results

MDSC are elevated with a concomitant decrease in CD8+ T cells during progression of lung cancer

For delineating the role of MDSC in progression of lung cancer, a murine lung cancer model was established by intravenous injection of LLC cells (106 cells) in syngeneic C57BL/6 mice. A significant increase in tumor burden was observed by 15 days following in vivo establishment and progression of lung cancer (Fig. 1A and B; P < 0.001 compared with early-stage tumor burden).

Recruitment of MDSCs were increased, whereas the infiltration of CD8+ and CD4+ T cells were decreased with tumor progression. A, tumor weights from mice on days 5, 10, 15, and 19 after intravenous challenge with 106 LLC tumor cells. **, P < 0.001 in comparisons with day 5 and 10 compared with day 5 and 15 compared with day 10 and 19 compared with day 15 (n = 5 mice/time point, three replicate experiments). B, hematoxylin and eosin staining of lung tissue at indicated time points. C, FACS plots showing percentages of MDSC in tumor on days 10, 15, and 19 post-LLC injection, P < 0.01 for Gr-1+CD11b+ MDSC at day 19 versus day 15 and for day 10 versus day 15. D, characterization of MDSC subsets by flow cytometry using additional MDSC markers Ly-6C, Ly-6G, and F4/80. E, FACS plots showing CD8+ and CD4+ T cells in tumor at indicated times. Left to right, P < 0.05 for both CD4+ and CD8+ T cells, day 10 versus day 15 versus day 19 (n = 5 mice/time point, three replicate experiments). The percentages displayed on FACS plots are from a representative run.

We first investigated the progression of tumor growth in the lungs and the significance of infiltrating immunosuppressive cells in the TME. Enumeration of immune cell phenotypes by flow cytometry demonstrated an increase in tumor-infiltrating MDSC with increasing tumor growth (Fig. 1C). The CD11bintGr-1int MDSC population stained positive for both Ly-6C and F4/80 (markers characteristic of monocytic phenotype of MDSC), whereas the CD11bhiGr-1hi MDSC population expressed both Ly-6G and F4/80 (markers characteristic of granulocytic phenotype of MDSC; Fig. 1D). These MDSC subsets were also characterized in lung and spleen (Supplementary Fig. S1). As the numbers of MDSC increased with tumor burden, a significant reduction in CD8+ and CD4+ T cells was observed (Fig. 1E, same time points as Fig. 1C; P < 0.05 with increased tumor growth). Similar enhanced infiltration of MDSC and a steady decline in CD8+ T cells with tumor progression was also noted following intracardiac implantation of tumor cells (Supplementary Fig. S1C).

Treatment of tumor-bearing mice with gemcitabine and a SOD mim targets MDSC and reduces tumor progression

A significant reduction in tumor-infiltrating MDSC numbers was noted following combination therapy compared with all other treatment groups (Fig. 2C; P < 0.01), with similar observations in lung and spleen tissues (Supplementary Fig. S2A). Furthermore, ROS levels associated with MDSC and other ROS-contributing immune cell types including tumor-associated macrophages (TAM) and tumor-associated neutrophils (TAN) were significantly reduced in the combination therapy group as compared with the PBS control group (Fig. 2D, P < 0.01 for ROS+ MDSC and non-MDSC cells), reflecting an overall reduction of total ROS in TME. Although we observed a significant reduction in tumor-infiltrating neutrophils (Supplementary Fig. S3A), macrophage infiltration was not modulated by combination therapy.

MDSC may also induce development and migration of Tregs to the TME, which can then inhibit antitumor responses and contribute to immunologic tolerance in cancer (18). As shown in Fig. 2E, combination therapy of SOD mim + Gem also significantly reduced the infiltration of Treg (P < 0.05).

Combination therapy with a SOD mim and gemcitabine enhances the CD8+ T-cell response

ROS-mediated inhibition of CD8+ T-cell response is the primary immunosuppressive mechanism of MDSC (19, 20). Because increased MDSC infiltration was associated with a reduction of CD8+ T cells during tumor progression, we investigated whether depleting MDSC with combination therapy would modulate the CD8+ T-cell response. The total percentages and absolute numbers of CD8+ T cells increased in tumor, lung, and spleen tissues of mice treated with SOD mim + Gem (Fig. 3A and B) Immunohistochemical analysis further showed a significant increase in the infiltration of CD8+ T cells and a decrease in infiltration of Gr-1+ cells in the periphery as well as center of tumor tissue in mice treated with combination therapy as compared with controls (Fig. 3C). Furthermore, the combination therapy significantly reduced the percentages of apoptotic Annexin+ CD8+ T cells (Fig. 3D; P < 0.001 in comparison with all other treatments and control groups).

SOD mim + Gem treatment results in enhanced CD8+ T-cell recruitment and diminished presence of MDSC. A, the percentage of CD8+ T cells was determined by flow-cytometric analyses of cells harvested from lung, spleen, and tumor tissues. Live CD3+ cells in the lymphocyte gate were analyzed for percentage of CD8+ cells. **, P < 0.01 when compared with all treatment groups as derived by ANOVA. B, total numbers of CD8+ T cells in lung, spleen, and tumor tissues from each treatment group are presented. C, localization of Gr-1+ and CD8+ cells as determined by immunofluorescent analyses of OCT frozen tumor tissue stained with anti-Gr-1 and CD8 antibodies. D, percentage Annexin+ CD8+ T cells as determined by flow cytometry of cells harvested from lung, spleen, and tumor tissues. **, P < 0.001 in comparison with all other treatment and control groups.

Combination therapy enhances the memory CD8+ T-cell response

The effectiveness of CD8+ T cells to tumor challenge is dependent on the state of their differentiation. We investigated whether the increased CD8+ T-cell viability and cell numbers following targeted depletion of MDSC and ROS pathways reflected an increased differentiation into various effector and memory subsets. As shown in Fig. 4A, a significant and rapid increase in the percentages of effector (TEM), central (TCM), and stem cell (TSCM) memory CD8+T cells was observed with tumor progression (P < 0.001 for SOD mim + Gem vs. Gem for TEM and TSCM). At 14 days after tumor establishment and 6 to 7 days after therapy, TEM and TSCM subsets were observed in increased numbers. However, the pool of CD8+ memory subsets changed 72 hours later, with significantly higher percentages of all three subsets (Fig. 4B; P < 0.01 for SOD mim + Gem vs. Gem for TEM and TSCM; P < 0.05 for the same comparison for TCM). Increased TEM levels were also observed in the group treated with only gemcitabine. Further characterization of CD8+ T cells indicated that they expressed stem cell antigen-1 (Sca-1; Fig. 4C; P < 0.001 for SOD mim + Gem compared with Gem and PBS; P < 0.01 for SOD mim vs. Gem; ref. 21), a marker found on self-renewing CD8+ T cells capable of generating central and effector memory populations. Coexpression of high CD62L expression defined these cells as TSCM as shown in Fig. 4D. The combination therapy, however, did not directly affect the viability of CD4+ or CD8+ T cells ex vivo (data not shown). Enhanced CD8+ memory T-cell response was also noted following anti-Gr-1 monoclonal antibody (mAb)–mediated depletion of MDSC and targeting MDSC function with SOD mim (Supplementary Fig. S3B). We did not observe any enhanced targeting effects of SOD mim on LLC tumor cells in vitro as pretreatment of LLC tumor cells with increasing concentrations of SOD mim did not sensitize the cells further to treatment with gemcitabine (Supplementary Fig. S3C). Combination treatment also reduced MDSC infiltration and ROS levels in the TME of catalase-deficient mice, which normally have elevated ROS levels. In addition, it reduced the tumor burden (Supplementary Fig. S4A and S4D) and enhanced the memory response (increased percentages of TEM, TCM, and TSCM) to levels noted in the WT mice (Supplementary Fig. S4B and S4C).

Reduction of ROS levels is associated with reduced thiol groups on T-cell surfaces, which contributes to persistence of memory cells and protection from apoptotic cell death (22). TCM are reported to have more reduced thiols as compared with TEM and can withstand oxidative stress (22, 23). Therefore, we hypothesized that CD8+ T cells purified from the combination therapy treated mice may express high levels of thiols and therefore, are more effective in antitumor response. On the basis of maleimide reactivity, an increase in reduced thiols was detected in CD8+ T cells obtained from tumor, spleen, and lungs of mice treated with combination therapy (Fig. 5A; P < 0.01 for SOD mim + Gem vs. Gem). Furthermore, phosphorylation of a thiol-dependent transcription factor STAT-3 (24, 25), critical for memory CD8+ T-cell function (26), was in memory cells following combination therapy. As demonstrated by phospho-flow analysis of memory subsets (Fig. 5B), increased levels of phosphorylated STAT-3 were detected in tumor-derived TEM and TCM obtained from mice given combination therapy when compared with gemcitabine therapy alone. In addition, the ratio of p-STAT-3 to total STAT-3 changed significantly in the memory cells as compared with effector cells and the percentage of phosphorylation was enhanced in the combination therapy group as compared with all the other individual treatments (Fig. 5C; P < 0.05 for SOD mim + Gem vs. Gem for TEM and TCM). We then investigated whether the combination therapy triggers switching of the metabolic pathways that fuel the energy production required for function of the memory T-cell subsets. LC/MS-MRM analysis of metabolites of these CD8+ T-cell memory subsets (Fig. 5D) indicated that both TEM and TCM subsets from combination therapy had a higher dependency on glycolysis compared with those from individual therapies alone. Thus, the reduction of MDSC infiltration and MDSC-associated ROS altered the metabolic status of these tumor-specific CD8+ T-cell memory subsets.

We then investigated whether the quality of the CD8+ T-cell response is modulated by the combination therapy. We first compared the cytotoxic potential of these tumor-specific memory CD8+ T-cell subsets. As shown in Fig. 5E, the cytolytic activity of both TEM and TCM derived from tumor tissues of SOD mim + Gem therapy mice was more potent against target lung cancer cells when compared with those obtained from mice receiving individual therapies (P < 0.05). The TCM purified from the SOD mim therapy group were also cytolytic. Importantly, the CD8+ T cells purified from mice treated with combination therapy showed an enhancement of the polyfunctional response with increased percentages of IFN-γ, cytoplasmic perforin, and IL-2+ CD8+ T cells (Fig. 6A–C; P < 0.01 for IFN-γ expression; P < 0.001 for perforin+ CD8+T cells, and IL-2+ CD8+ T cells from tumor). Thus, it is likely that the central role of establishing memory response following combination therapy was not only for long-term antitumor immune senescence but also against relapse following long-term remission.

Because the increased infiltration of the memory CD8+ T cells following combination therapy lowered lung cancer progression, we investigated whether adoptive transfer of these memory cells would improve survival of mice with established lung tumors. For these studies, effector, TCM, and TEM CD8+ T cells were purified from gemcitabine only and SOD mim + Gem therapy groups and were adoptively transferred into tumor-bearing mice 7 days after lung cancer challenge (Fig. 7A). As shown in Fig. 7B, 80% of mice that received either TCM or TEM from the SOD mim + Gem group survived for up to 80 days after lung cancer challenge as compared with 25-day mean survival of treated mice in control groups (P < 0.001 for G+S-E CD8, G+S-TCM CD8, G+S-TEM CD8 and Gem-TEM CD8 compared with PBS group; P < 0.05 for PBS group when compared with Gem-E CD8).

Mice that survived for up to 80 days postadoptive transfer were rechallenged with LLC cells to investigate the efficacy of these adoptively transferred memory subsets and their potential to be reactivated in the presence of tumor antigens. Enumeration of circulating memory CD8+ T memory subsets showed a significant increase in the TSCM population in rechallenged mice as compared with the baseline levels (levels of memory cells in adoptive transferred mice before lung cancer rechallenge) and in comparison with tumor challenged control mice (tumor bearing mice without CD8+ T-cell adoptive transfer; Fig. 7C and Supplementary Fig. S5; P < 0.001 for TSCM and TCM in rechallenged groups compared with baseline and controls). These persistent memory CD8+ T-cell subsets were efficient in maintaining reduced tumor burden. A reduction in infiltration of MDSC levels was also noted in the adoptive transfer recipients (Fig. 7D and Supplementary Fig. S6; P < 0.05 and P < 0.01 for TCM compared with TEM and control, respectively). In addition to the increased memory subsets in circulation, congenic adoptive transfer of memory subsets further confirmed the expansion of adoptively transferred CD8+ T-cell memory subsets specifically in the tumor tissue (Supplementary Fig. S7).

Collectively, this study convincingly demonstrates that targeting the proliferation and immunosuppressive functions of MDSC promote antitumor immunity by enhancing the quantity and quality of the CD8+ T-cell responses and promote persistent immunologic memory, thereby reducing tumor burden and prolonging survival of mice with lung cancer.

Discussion

Recent evidence suggests that immune regulation contributed by MDSC in the TME dampens the long-term efficacy of existing combination chemotherapies for lung cancer (10). ROS produced by MDSC contributes to immunosuppression in the TME (7, 8). MDSC also replenish the tumor stroma with precursors of both TAM and TAN, which contribute to oxidative stress in the TME (27). The multifaceted impact of oxidative stress in the TME is reflected not only in the impairment of T cell and natural killer (NK) cell activity, but also on redox-mediated regulation of T-cell signaling and T-cell survival (22, 28). The differentiation state of the lymphocytes determines their function and persistence (29, 30). Mechanisms that modulate survival and function of T-cell subsets, particularly in regard to their differential sensitivity to oxidative stress in the TME, have yet to be completely elucidated. This is of great relevance in the context of memory CD8+ T cells, which are a critical component of protective immunity against cancer. Differential sensitivity of memory cell phenotypes to oxidative stress may skew the memory repertoire to a particular subset, leading to its persistence in TME. Therefore, strategies that eliminate MDSC-mediated immunosuppression in combination with antioxidant therapeutics to enhance the CD8+ T-cell effector and memory response may be efficacious as novel anti-lung cancer regimens.

In this study, we provide evidence that gemcitabine when used in combination with an SOD mim, result in successful inhibition of MDSC, Treg, and TAN infiltration and a significant reduction of ROS in the TME (Fig. 2). Inhibition of MDSC observed with this therapy is supported by recent studies that demonstrate efficacy of chemotherapeutic strategies in targeting MDSC (31–33). The observed reduction of infiltration of Treg is consistent with depletion of MDSC, as MDSCs induce development (34) and promote migration and recruitment of Treg to sites of chronic inflammation (35). The reduction in neutrophils observed in the tumor tissue following combination therapy is consistent with the reduction in MDSC, as MDSC infiltration have been associated with increased TAN in the TME. The enhanced percentages and absolute numbers of CD8+ T cells and reduction in their apoptotic death following combination therapy (Fig. 3) suggest a potential utility of this approach for reducing tumor burden and stimulating antitumor immunity by providing an ideal microenvironment for optimal T-cell function.

Our novel observation that the combination therapy enhances both the quantity and quality of the memory response with polyfunctional and cytolytic activity, all support the potential of this strategy to promote T-cell survival and function (Figs. 4 and 5). In an MDSC- and ROS-depleted environment, the memory response to tumor challenge were skewed toward TCM, TEM, and more interestingly, a subset TSCM with a stem cell memory phenotype (Fig. 4). Similar observations noted with MDSC depletion using anti-Gr-1 Ab and SOD mim further validates these findings. TSCM CD8+ T cells have extensive replicative potential in vivo and maintain the naïve CD44−CD62L+ phenotype (30). These cells display the glycosylphosphatidyl inositol-linked molecule Sca-1, a marker for self-renewing cells (22, 29). Our studies showed a significant early increase in TEM and TSCM phenotypes followed by a shift in the expansion of both TCM and TSCM (Fig. 4) as a late response to combination therapy. It is possible that redox regulation by MDSC may cause an intrinsic defect in the ability of TEM and TCM to proliferate in response to tumor challenge and this defect could be reversed or rectified by depletion of MDSC and associated ROS in the TME. Oxidative stress in the TME may also affect the differentiation pathways of these memory subsets and skew the memory repertoire toward subsets that are less sensitive to oxidative stress such as the TCM and TEM (22, 28). Depletion of ROS and MDSC from TME could then trigger the expansion of these resistant memory cell subsets, as well as TSCM (stem cell phenotype with high proliferative capacity). Consistent with these possibilities, our data suggest that combination therapy-based depletion of the excess ROS and MDSC enhanced the CD8+ T-cell memory response, consistently skewing the memory repertoire to TCM and TEM cells, and increased numbers of TSCM (Fig. 4). These observations were also noted in catalase-deficient mice (Supplementary Fig. S4). Our studies do not, however, delineate whether alterations in differentiation pathways of these T-cell subsets account for the observed cellular redistribution in the memory compartment.

SOD mims are effective free radical scavengers in many disease models including those of stroke (36), diabetes (37), and sickle cell anemia (38). They have been shown to protect normal tissues against radiation-induced damage presumably by alleviating oxidative stress (39). In addition, there is mounting evidence that SOD mims may enhance tumor radioresponsiveness (40, 41) Although the SOD mim, which we have used in our combination therapy, does not enhance tumor responsiveness to chemotherapy, it offers significant advantages for adoptive cell therapy strategies as it enhances persistent antitumor immunity.

Our data suggest that modulation of redox regulation and signaling by gemcitabine and combination therapy may impact long-lived memory populations. We show that lowered oxidative stress in the TME, resulting from depletion of MDSC and ROS, is associated with an increased number of cell surface thiols on TCM (Fig. 5). This is consistent with earlier reports that memory subsets resistant to activation induced cell death or apoptosis have increased thiols contributing to their persistence (22, 29). We do not anticipate any direct effects of SOD mim on thiols, but rather the combination therapy modulates upstream events, resulting in a change in the redox status of the memory cells. The modulation of reduced thiols on T-cell surface may reduce the threshold for T-cell activation and enhanced their proliferative capacity in vivo. The observed increase in phosphorylation of thiol-dependent STAT-3 in TCM as compared with TEM (Fig. 5B and C), may allow them to persist as memory cells, as STAT-3 activation is critical for maintenance of long-term memory (26).

Because metabolism underlies the functional capacity of T cells, their maintenance and their persistence during an immune response, therapeutic strategies that manipulate the T-cell metabolism may alter the outcome of the antitumor response (42). This is particularly relevant in our observations that the combination therapy not only enhanced the CD8+ memory T-cell response, but also modulated the metabolism of TCM and TEM subsets. These memory cells were glycolysis-dependent and relied on a more readily available ATP source than those resulting from individual therapies (Fig. 5D). These observations are also consistent with the association of STAT-3 activation and persistence of TCM and TEM subsets in adoptive transfers and rechallenges with tumor cells. TCM and TEM subsets from combination therapy responded quickly and vigorously to the tumor rechallenge compared with controls. Adoptive transfer recipients of TCM purified from combination therapy–treated mice were more efficient than TEM in eradicating tumor burden and prolonging survival (Fig. 7). In addition, rechallenging the surviving animals with a second round of tumor cells produced an increased expansion of TCM as compared with TEM, successful eradication of tumor burden, and a reduced infiltration of MDSC (Fig. 7). We have not delineated, however, whether the changes in the metabolic program of CD8+ memory T-cell subsets triggered by combination therapy is to either increase energy production to cope with the increased demand to proliferate, to provide biosynthetic precursors or to generate reducing equivalents to balance the change in redox status in the TME.

We have not investigated the efficacy of these memory cells after repeated exposure to the antigen or their efficiency in targeting tumor cells following repeated cycles of adoptive transfer. In addition, we have not addressed the potential of this strategy for small cell lung cancer, cancers other than lung cancers, and/or other cancers that metastasize to the lung. Nevertheless, our investigations certainly suggest that depletion of MDSC and ROS may provide a preferential advantage for TCM to be more functionally active, proliferative, and persistent to provide long-term immunity against lung cancer.

This is particularly relevant for adoptive cell transfer therapies that are being tested in early-stage clinical trials for advanced cancer. There is also an impending need for novel effective approaches to generate persistent immunity in patients in whom the potential for complete elimination of tumor antigens are highly unlikely. Therefore, defining determinants for successful CD8+ T-cell adoptive immunotherapy are essential. Our combination therapy offers an attractive strategy for adoptive T-cell therapy in that it has potential to generate long-lived populations of TCM equipped with the dual potential of both immune surveillance, as well as tumor eradication. In addition, this therapeutic strategy enhances memory cells with a self-renewing phenotype that may increase their long-term efficacy and persistence. We believe there is a potential role for this combination therapy as an adjuvant therapy in treatment strategies to eradicate early lung cancer and has implications for prevention of lung cancer relapse or recurrence.

Grant Support

This study was supported by the Collaborative Development Grant for Immunology and Cancer Immunotherapeutics funded by the National Cancer Institute (NCI CA13148-39 to J.S. Deshane and S. Ponnazhagan), R01CA132077 and R01CA133737 to S. Ponnazhagan. Funds for the operation of the Targeted Metabolomics and Proteomics Laboratory come in part from the UAB Center for Nutrient-Gene Interaction (U54 CA 100949), the Purdue-UAB Botanicals Center for Age-Related Disease (P50 AT00477), the UAB O'Brien Acute Kidney Injury Center (P30 DK079337), the UAB Skin Disease Research Center (P30 AR50948), and the UAB Lung Health Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

The authors thank Doyle Ray Moore and Dr. Stephen Barnes of the Targeted Metabolomics and Proteomics Laboratory, UAB for the metabolomic analyses included in this study. The authors also thank Marion Spell (Center for AIDS Research Flow Facility Core for cell sorting) and Enid Keyser (Rheumatic Diseases Analytic and Preparative Cytometry Facility) for their technical support for fluorescence-activated cell sorting (FACS).

Footnotes

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).